Human respiratory syncytial virus (RSV) outranks all other microbial pathogens as a cause of pneumonia and bronchiolitis in infants under one year of age. Virtually all children are infected by two years of age, and reinfection occurs with appreciable frequency in older children and young adults (Chanock et al., in Viral Infections of Humans, 3rd ed., A. S. Evans, ed., Plenum Press, N.Y. (1989). RSV is responsible for more than one in five pediatric hospital admissions due to respiratory tract disease, and causes an estimated 91,000 hospitalizations and 4,500 deaths yearly in the United States alone. Although most healthy adults do not have serious disease due to RSV infection, elderly patients and immunocompromised individuals often suffer severe and possibly life-threatening infections from this pathogen.
Despite decades of investigation to develop effective vaccine agents against RSV, no safe and effective vaccine has yet been achieved to prevent the severe morbidity and significant mortality associated with RSV infection. Failure to develop successful vaccines relates in part to the fact that small infants have diminished serum and secretory antibody responses to RSV antigens. Thus, these individuals suffer more severe infections from RSV, whereas cumulative immunity appears to protect older children and adults against more serious impacts of the virus. One antiviral compound, ribavarin, has shown promise in the treatment of severely infected infants, although there is no indication that it shortens the duration of hospitalization or diminishes the infant""s need for supportive therapy.
The mechanisms of immunity in RSV infection have recently come into focus. Secretory antibodies appear to be most important in protecting the upper respiratory tract, whereas high levels of serum antibodies are thought to have a major role in resistance to RSV infection in the lower respiratory tract. Purified human immunoglobulin containing a high titer of neutralizing antibodies to RSV may prove useful in some instances of immunotherapeutic approaches for serious lower respiratory tract disease in infants and young children. Immune globulin preparations, however, suffer from several disadvantages, such as the possibility of transmitting blood-borne viruses and difficulty and expense in preparation and storage.
RSV-specific cytotoxic T cells, another effector arm of induced immunity, are also important in resolving an RSV infection. However, while this latter effector can be augmented by prior immunization to yield increased resistance to virus challenge, the effect is short-lived. The F and G surface glycoproteins are the two major protective antigens of RSV, and are the only two RSV proteins which have been shown to induce RSV neutralizing antibodies and long term resistance to challenge (Collins et al., Fields Virology, Fields et al. eds., 2:1313-1352. Lippincott-Raven, Philadelphia. (1996); Connors et al., J. Virol. 65(3):1634-7 (1991)). The third RSV surface protein, SH, did not induce RSV-neutralizing antibodies or significant resistance to RSV challenge.
One obstacle to development of live RSV vaccines is the difficulty in achieving an appropriate balance between attenuation and immunogenicity. Genetic stability of attenuated viruses also can be a problem. Vaccine development also is impeded by the relatively poor growth of RSV in cell culture and the instability of the virus particle. Another feature of RSV infection is that the immunity which is induced is not fully protective against subsequent infection. A number of factors probably contribute to this, including the relative inefficiency of the immune system in restricting virus infection on the luminal surface of the respiratory tract, the short-lived nature of local mucosal immunity, rapid and extensive virus replication, reduced immune responses in the young due to immunological immaturity, immunosuppression by transplacentally derived maternal serum antibodies, and certain features of the virus such as a high degree of glycosylation of the G protein. Also, as will be described below, RSV exists as two antigenic subgroups A and B, and immunity against one subgroup is of reduced effectiveness against the other.
Although RSV can reinfect multiple times during life, reinfections usually are reduced in severity due to protective immunity induced by prior infection, and thus immunoprophylaxis is feasible. A live-attenuated RSV vaccine would be administered intranasally to initiate a mild immunizing infection. This has the advantage of simplicity and safety compared to a parenteral route. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. It also abrogates the immunosuppressive effects of RSV-specific maternally-derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications (Murphy et al., Vaccine 8(5):497-502 (1990)), this has never been observed with a live virus.
Formalin-inactivated virus vaccine was tested against RSV in the mid-1960s, but failed to protect against RSV infection or disease, and in fact exacerbated symptoms during subsequent infection by the virus. (Kim et al., Am. J. Epidemiol., 89:422-434 (1969), Chin et al., Am J. Epidemiol., 89:449-463 (1969); Kapikian et al., Am. J. Epidemiol., 89:405-421 (1969)).
More recently, vaccine development for RSV has focused on attenuated RSV mutants. Friedewald et al., J. Amer. Med. Assoc. 204:690-694 (1968) reported a cold passaged mutant of RSV (cpRSV) which appeared to be sufficiently attenuated to be a candidate vaccine. This mutant exhibited a slight increased efficiency of growth at 26xc2x0 C. compared to its wild-type (wt) parental virus, but its replication was neither temperature sensitive nor significantly cold-adapted. The cold-passaged mutant, however, was attenuated for adults. Although satisfactorily attenuated and immunogenic for infants and children who had been previously infected with RSV (i.e., seropositive individuals), the cpRSV mutant retained a low level virulence for the upper respiratory tract of seronegative infants.
Similarly, Gharpure et al., J. Virol. 3:414-421 (1969) reported the isolation of temperature sensitive RSV (tsRSV) mutants which also were promising vaccine candidates. One mutant, ts-1, was evaluated extensively in the laboratory and in volunteers. The mutant produced asymptomatic infection in adult volunteers and conferred resistance to challenge with wild-type virus 45 days after immunization. Again, while seropositive infants and children underwent asymptomatic infection, seronegative infants developed signs of rhinitis and other mild symptoms. Furthermore, instability of the ts phenotype was detected, although virus exhibiting a partial or complete loss of temperature sensitivity represented a small proportion of virus recoverable from vaccinees, and was not associated with signs of disease other than mild rhinitis.
These and other studies revealed that certain cold-passaged and temperature sensitive RSV strains were underattenuated and caused mild symptoms of disease in some vaccinees, particularly seronegative infants, while others were overattenuated and failed to replicate sufficiently to elicit a protective immune response, (Wright et al., Infect. Immun., 37:397-400 (1982)). Moreover, genetic instability of candidate vaccine mutants has resulted in loss of their temperature-sensitive phenotype, further hindering development of effective RSV vaccines. See generally, Hodes et al., Proc. Soc. Exp. Biol. Med. 145:1158-1164 (1974), McIntosh et al., Pediatr. Res. 8:689-696 (1974), and Belshe et al., J. Med. Virol., 3:101-110 (1978).
Abandoning the approach of creating suitably attenuated RSV strains through undefined biological methods such as cold-passaging, investigators tested subunit vaccine candidates using purified RSV envelope glycoproteins. The glycoproteins induced resistance to RS virus infection in the lungs of cotton rats, Walsh et al., J. Infect. Dis. 155:1198-1204 (1987), but the antibodies had very weak neutralizing activity and immunization of rodents with purified subunit vaccine led to disease potentiation (Murphy et al., Vaccine 8:497-502(1990)).
Vaccinia virus recombinant-based vaccines which express the F or G envelope glycoprotein have also been explored. These recombinants express RSV glycoproteins which are indistinguishable from the authentic viral counterpart, and rodents infected intradermally with vaccinia-RSV F and G recombinants developed high levels of specific antibodies that neutralized viral infectivity. Indeed, infection of cotton rats with vaccinia-F recombinants stimulated almost complete resistance to replication of RSV in the lower respiratory tract and significant resistance in the upper tract. Olmsted et al., Proc. Natl. Acad. Sci. USA 83:7462-7466 (1986). However, immunization of chimpanzees with vaccinia-F and -G recombinant provided almost no protection against RSV challenge in the upper respiratory tract (Collins et al., Vaccine 8:164-168 (1990)) and inconsistent protection in the lower respiratory tract (Crowe et al., Vaccine 11:1395-1404 (1993).
The unfulfilled promises of attenuated RSV strains, subunit vaccines, and other strategies for RSV vaccine development underscores a need for new methods to develop novel RSV vaccines, particularly methods for manipulating recombinant RSV to incorporate genetic changes to yield new phenotypic properties in viable, attenuated RSV recombinants. However, manipulation of the genomic RNA of RSV and other negative-sense RNA viruses has heretofore proven difficult. Major obstacles in this regard include non-infectivity of naked genomic RNA of these viruses, poor viral growth in tissue culture, lengthy replication cycles, virion instability, a complex genome, and a refractory organization of gene products.
Recombinant DNA technology has made it possible to recover infectious negative-stranded RNA viruses from cDNA, to genetically manipulate viral clones to construct novel vaccine candidates, and to rapidly evaluate their level of attenuation and phenotypic stability (for reviews, see Conzelmann, J. Gen. Virol. 77:381-89 (1996); Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, (1996)). In this context, recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), parainfluenza virus (PIV), rabies virus (RaV), vesicular stomatitis virus (VSV), measles virus (MeV), and Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094 (1995); Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81 (1995); Radecke et al., EMBO J. 14:5773-5784 (1995); Schnell et al., EMBO J. 13:4195-203 (1994); Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92 (1995); Hoffman et al., J Virol. 71:4272-4277 (1997); Kato et al., Genes to Cells 1:569-579 (1996), Roberts et al., Virology 247(1), 1-6 (1998); Baron et al., J Virol. 71:1265-1271 (1997); International Publication No. WO 97/06270; Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567 (1995); Durbin et al., Virology 235:323-332 (1997); U.S. patent application Ser. No. 08/892,403, filed Jul. 15, 1997 (corresponding to published International Application No. WO 98/02530 and priority U.S. Provisional Application Nos. 60/047,634, filed May 23, 1997, 60/046,141, filed May 9, 1997, and 60/021,773, filed Jul. 15, 1996); Juhasz et al., J. Virol. 71(8):5814-5819 (1997); He et al. Virology 237:249-260 (1997); Baron et al. J. Virol. 71:1265-1271 (1997); Whitehead et al., Virology 247(2):232-9 (1998a); Whitehead et al., J. Virol. 72(5):4467-4471 (1998b); Jin et al. Virology 251:206-214 (1998); Bucholz et al. J. Virol. 73:251-259 (1999); and Whitehead et al., J. Virol. 73:(4)3438-3442 (1999), each incorporated herein by reference).
Among the remaining challenges to RSV vaccine development is the difficulty of achieving vaccine candidates that are effective against a broad range of existing and emergent strains and subgroups of RSV. In particular, it will be useful to provide a RSV subgroup B-specific vaccine virus, as well as multivalent vaccines to provide protection against both RSV A and RSV B subgroups. In this context, recent research has focused on development of chimeric viruses to carry antigenic determinants between viral strains. For example, the HN and F glycoproteins of human parainfluenza virus type 3 (PIV3) have been replaced by those of human parainfluenza virus type 1 (HPIV1), and the resulting chimeric virus grew in cell culture and in experimental animals with an efficiency similar to its wild-type parents (Tao et al., J. Virol. 72(4):2955-61 (1998), incorporated herein by reference. Also reported is a chimeric measles virus where the H and F glycoproteins were replaced with the G glycoprotein of vesicular stomatitis virus, which was inserted with or without replacement of the cytoplasmic and transmembrane region of G with that of measles virus F (Spielhofer et al., J. Virol. 72(3):2150-9 (1998)). This yielded a chimeric virus that was reportedly 50-fold reduced in growth. In a third example, Jin et al., Virology 251(1):206-14 (1998) report a subgroup A virus which expresses the G protein of a subgroup B RSV as an additional gene (Jin et al., Virolovy 251(1):206-14 (1998)). However, since the F protein also exhibits significant subgroup-specificity, it would be preferable to express both subgroup B glycoproteins in a subgroup B-specific vaccine. In addition, production of a chimeric A-B virus will not produce a viable vaccine candidate without further modifications to achieve proper attenuation and virulence.
Accordingly, an urgent need remains in the art for tools and methods to engineer safe and effective vaccines to alleviate the serious health problems attributable to RSV, particularly that will be effective against multiple existing and emergent strains and subgroups of RSV. Quite surprisingly, the present invention satisfies these and other related needs.
The present invention provides chimeric, recombinant respiratory syncytial virus (RSV) that are infectious and elicit a propylactic or therapeutic immune response in humans or other mammals. In related aspects, the invention provides novel methods and compositions for designing and producing attenuated, chimeric RSV suitable for vaccine use. Included within these aspects of the invention are novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a chimeric RSV genome or antigenome including a partial or complete RSV genome or antigenome of one RSV strain or subgroup virus combined with one or more heterologous gene(s) or gene segment(s) of a different RSV strain or subgroup virus. Also provided within the invention are methods and compositions-incorporating chimeric, recombinant RSV for prophylaxis and treatment of RSV infection.
Chimeric RSV of the invention are recombinantly engineered to incorporate nucleotide sequences from more than one RSV strain or subgroup to produce an infectious, chimeric virus or subviral particle. In this manner, candidate vaccine viruses are recombinantly engineered to elicit an immune response against RSV in a mammalian host susceptible to RSV infection, including humans and non-human primates. Chimeric RSV according to the invention may elicit an immune response to a specific RSV subgroup or strain, or a polyspecific response against multiple RSV subgroups or strains.
Exemplary chimeric RSV of the invention incorporate a chimeric RSV genome or antigenome, as well as a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large polymerase protein (L), and a RNA polymerase elongation factor. Additional RSV proteins may be included in various combinations to provide a range of infectious subviral particles as well as complete viral particles.
Chimeric RSV of the invention include a partial or complete RSV genome or antigenome from one RSV strain or subgroup virus combined with one or more heterologous gene(s) or gene segment(s) of a different RSV strain or subgroup virus to form the chimeric RSV genome or antigenome. In preferred aspects of the invention, chimeric RSV incorporate a partial or complete human RSV genome or antigenome of one RSV subgroup or strain combined with one or more heterologous gene(s) or gene segment(s) from a different human RSV subgroup or strain. For example, a chimeric RSV may incorporate a chimeric genome or antigenome comprised of a partial or complete human RSV A subgroup genome or antigenome combined with one or more heterologous gene(s) or gene segment(s) from a human RSV B subgroup virus.
Heterologous genes or gene segments from one RSV strain or subgroup represent xe2x80x9cdonorxe2x80x9d genes or polynucleotides that are combined with, or substituted within, a xe2x80x9crecipientxe2x80x9d genome or antigenome. The recipient genome or antigenome typically acts as a xe2x80x9cbackbonexe2x80x9d or vector to import heterologous genes or gene segments to yield a chimeric RSV exhibiting novel phenotypic characteristics. For example, addition or substitution of heterologous genes or gene segments within a selected recipient RSV strain may result in attenuation, growth changes, altered immunogenicity, or other desired phenotypic changes as compared with a corresponding phenotype(s) of the unmodified recipient and/or donor. Genes and gene segments that may be selected for use as heterologous inserts or additions within the invention include genes or gene segments encoding a NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F or G protein or portion thereof.
In preferred embodiments of the invention, chimeric RSV incorporates one or more heterologous gene(s) that encode an RSV F, G or SH glycoprotein. Alternatively, the chimeric RSV may incorporate a gene segment encoding a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope of a RSV F, G or SH glycoprotein. These immunogenic proteins, domains and epitopes are particularly useful within chimeric RSV because they can generate novel immune responses in an immunized host.
For example, addition or substitution of one or more immunogenic gene(s) or gene segment(s) from one donor RSV subgroup or strain within a recipient genome or antigenome of a different RSV subgroup or strain can generate an immune response directed against the donor subgroup or strain or against both the donor and recipient subgroup or strain. In one exemplary embodiment, one or more human RSV subgroup B glycoprotein genes F, G and SH or a cytoplasmic domain, transmembrane domain, ectodomain or immunogenic epitope thereof, is added to, or substituted within, an RSV A genome or antigenome.
In additional aspects of the invention, attenuated, chimeric RSV are produced in which the chimeric genome or antigenome is further modified by introducing one or more attenuating point mutations specifying an attenuating phenotype. These point mutations may be generated de novo and tested for attenuating effects according to a rational design mutagenesis strategy. Alternatively, the attenuating point mutations are identified in biologically derived mutant RSV and thereafter incorporated into a chimeric RSV of the invention.
Preferably, chimeric RSV of the invention are attenuated by incorporation of at least one, and more preferably two or more, attenuating point mutations identified from a panel of known, biologically derived mutant RSV strains. Preferred mutant RSV strains described herein are cold passaged (cp) and/or temperature sensitive (ts) mutants, for example the mutants designated xe2x80x9ccpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579)xe2x80x9d (each deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., and granted the above identified accession numbers). From this exemplary panel of biologically derived mutants, a large xe2x80x9cmenuxe2x80x9d of attenuating mutations are provided which can each be combined with any other mutation(s) within the panel for calibrating the level of attenuation in the recombinant, chimeric RSV for vaccine use. Additional mutations may be derived from RSV having non-ts and non-cp attenuating mutations as identified, e.g., in small plaque (sp), cold-adapted (ca) or host-range restricted (hr) mutant strains.
In yet additional aspects of the invention, chimeric RSV, with or without attenuating point mutations, are mutated by a non-point nucleotide modification to produce desired phenotypic, structural, or functional changes. Typically, the selected nucleotide modification will specify a phenotypic change, for example a change in growth characteristics, attenuation, temperature-sensitivity, cold-adaptation, plaque size, host range restriction, or immunogenicity. Structural or functional changes include introduction or ablation of restriction sites into RSV encoding cDNAs for ease of manipulation and identification.
In preferred embodiments, an SH, NS1, NS2 or G gene is modified in the chimeric RSV, e.g., by deletion of the gene or ablation of its expression. Alternatively, the nucleotide modification can include a deletion, insertion, addition or rearrangement of a cis-acting regulatory sequence for a selected RSV gene.
In one example, a cis-acting regulatory sequence of one RSV gene is changed to correspond to a heterologous regulatory sequence, which may be a counterpart cis-acting regulatory sequence of the same gene in a different RSV or a cis-acting regulatory sequence of a different RSV gene. For example, a gene end signal may be modified by conversion or substitution to a gene end signal of a different gene in the same RSV strain.
In a separate embodiment, the nucleotide modification may comprise an insertion, deletion, substitution, or rearrangement of a translational start site within the chimeric genome or antigenome, e.g., to ablate an alternative translational start site for a selected form of a protein. In one example, the translational start site for a secreted form of the RSV G protein is ablated to modify expression of this form of the G protein and thereby produce desired in vivo effects.
Yet additional modifications may be made to the chimeric RSV genome or antigenome according to the invention, including modifications that introduce into the chimeric genome or antigenome a non-RSV molecule such as cytokine, a T-helper epitope, a restriction site marker, or a protein of a microbial pathogen capable of eliciting a protective immune response against the pathogen in a mammalian host. In one such embodiment, chimeric RSV are constructed that incorporate a gene or gene segment from a parainfluenza virus (PIV), for example a PIV HN or F glycoprotein or an immunogenic domain or epitope thereof.
Chimeric RSV designed and selected for vaccine use often have at least two and sometimes three or more attenuating mutations to achieve a satisfactory level of attenuation for broad clinical use. In one embodiment, at least one attenuating mutation occurs in the RSV polymerase gene (either in the donor or recipient gene) and involves one or more nucleotide substitution(s) specifying an amino acid change in the polymerase protein specifying an attenuation phenotype which may or may not involve a temperature-sensitive (ts) phenotype. Exemplary chimeric RSV in this context incorporate one or more nucleotide substitutions in the large polymerase gene L resulting in an amino acid change at amino acid Phe521, Gln831, Met1169, or Tyr1321, as exemplified by the changes, Leu for Phe521, Leu for Gln831, Val for Met1169 and Asn for Tyr1321. Other alternative amino acid assignments at this position can of course be made to yield a similar effect as the identified, mutant substitution. In this context, it is prefarable to modify the chimeric genome or antigenome to encode an alteration at the subject site of mutation that corresponds conservatively to the alteration identified in the mutant virus. For example, if an amino acid substitution marks a site of mutation in the mutant virus compared to the corresponding wild-type sequence, then a similar substitution should be engineered at the corresponding residue(s) in the recombinant virus. Preferably the substitution will involve an identical or conservative amino acid to the substitute residue present in the mutant viral protein. However, it is also possible to alter the native amino acid residue at the site of mutation non-conservatively with respect to the substitute residue in the mutant protein (e.g., by using any other amino acid to disrupt or impair the-identity and functio of the wild-type residue). In the case of mutations marked by deletions or insertions, these can be introduced as corresponding deletions or insertions into the recombinant virus, however the particular size and amino acid sequence of the deleted or inserted protein fragment can vary. Chimeric RSV of the invention may incorporate a ts mutation in any additional RSV gene besides L, e.g., in the M2 gene.
Preferably, two or more nucleotide changes are incorporated in a codon specifying an attenuating mutation, e.g., in a codon specifying a ts mutation, thereby decreasing the likelihood of reversion from an attenuated phenotype.
Attenuating mutations may be selected in coding portions of a donor or recipient RSV gene or in non-coding regions such as a cis-regulatory sequence. Exemplary non-coding mutations include single or multiple base changes in a gene start sequence, as exemplified by a single or multiple base substitution in the M2 gene start sequence at nucleotide 7605 (nucleotide 7606 in recombinant sequence).
In another aspect of the invention, compositions (e.g., isolated polynucleotides and vectors incorporating an RSV-encoding cDNA) and methods are provided for producing an isolated infectious chimeric RSV. Using these compositions and methods, infectious chimeric RSV are generated from a chimeric RSV genome or antigenome, a nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large (L) polymerase protein, and an RNA polymerase elongation factor. In related aspects of the invention, compositions and methods are provided for introducing the aforementioned structural and phenotypic changes into a recombinant chimeric RSV to yield infectious, attenuated vaccine viruses.
In one embodiment, an expression vector is provided which comprises an isolated polynucleotide molecule encoding a chimeric RSV genome or antigenome. Also provided is the same or different expression vector comprising one or more isolated polynucleotide molecules encoding N, P, L and RNA polymerase elongation factor proteins. The vector(s) is/are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious chimeric RSV particle or subviral particle.
The RSV genome or antigenome and the N, P, L and RNA polymerase elongation factor (preferably the product of the M2(ORF1) of RSV) proteins can be coexpressed by the same or different expression vectors. In some instances the N, P, L and RNA polymerase elongation factor proteins are each encoded on different expression vectors. The polynucleotide molecule encoding the chimeric RSV genome or antigenome can be a chimera of different.human RSV subgroups or strains, for example a polynucleotide containing sequences from a subgroup A RSV operably joined with sequences from a subgroup B RSV. Alternatively, the chimeric genome or antigenome can be a chimera of human and non-human (e.g., bovine or murine) RSV sequences. In yet another alternative aspect of the invention, the chimeric genome or antigenome can be a chimera of RSV and non-RSV sequences, for example a polynucleotide containing sequences from a human RSV operably joined with PIV sequences. The chimeric genome or antigenome can be further modified by insertion, rearrangement, deletion or substitution of one or more nucleotides, including point mutations, site-specific nucleotide changes, and changes involving entire genes or gene segments introduced within a heterologous donor gene or gene segment or the recipient, background genome or antigenome. These alterations typically specify one or more phenotypic change(s) in the resulting recombinant RSV, such as a phenotypic change that results in attenuation, temperature-sensitivity, cold-adaptation, small plaque size, host range restriction, alteration in gene expression, or a change in an immunogenic epitope.
The above methods and compositions for producing chimeric RSV yield infectious viral or subviral particles, or derivatives thereof. An infectious virus is comparable to the authentic RSV virus particle and is infectious as is. It can directly infect fresh cells. An infectious subviral particle typically is a subcomponent of the virus particle which can initiate an infection under appropriate conditions. For example, a nucleocapsid containing the genomic or antigenomic RNA and the N, P, L and M2(ORF1) proteins is an example of a subviral particle which can initiate an infection if introduced into the cytoplasm of cells. Subviral particles provided within the invention include, inter alia, viral particles which lack one or more protein(s), protein segment(s), or other viral component(s) not essential for infectivity.
Infectious chimeric RSV according to the invention can incorporate heterologous, coding or non-coding nucleotide sequences from any RSV or RSV-like virus, e.g., human, bovine, murine (pneumonia virus of mice), or avian (turkey rhinotracheitis virus) RSV, or from another enveloped virus, e.g., parainfluenza virus (PIV). In exemplary aspects, the recombinant RSV comprises a chimera of a human RSV genomic or antigenomic sequence recombinantly joined with one or more heterologous RSV sequence(s). Exemplary heterologous sequences include RSV sequences from one human RSV strain combined with sequences from a different human RSV strain. For example, chimeric RSV of the invention may incorporate sequences from two or more wild-type or mutant RSV strains, for example mutant strains selected from cpts RSV 248, cpts 248/404, cpts 248/955, cpts RSV 530, cpts 530/1009, or cpts 530/1030). Alternatively, chimeric RSV may incorporate sequences from two or more, wild-type or mutant RSV subgroups, for example a combination of RSV subgroup A and subgroup B sequences. In yet additional aspects, one or more human RSV coding or non-coding polynucleotides are substituted with a counterpart sequence from bovine or murine RSV, alone or in combination with one or more selected attenuating point mutations, e.g., cp and/or ts mutations, to yield novel attenuated vaccine strains. In one embodiment, a chimeric bovine-human RSV incorporates a substitution of the human RSV NP gene or gene segment with a counterpart bovine NP gene or gene segment, which chimera can optionally be constructed to incorporate a SH gene deletion, one or more cp or ts point mutations, or various combinations of these and other mutations disclosed herein.
In one embodiment of the invention, isolated polynucleotides, expression vectors, and methods for producing chimeric RSV are provided wherein the genome or antigenome is recombinantly altered compared to either the donor or recipient sequence. In particular, mutations are incorporated within a chimeric RSV genome or antigenome based on their ability to alter the structure and/or function of a chimeric RSV clone, e.g., by altering the structure, expression and or function of a selected protein encoded or a cis-acting RNA sequence thereby yielding a desired phenotypic change. Desired phenotypic changes include, e.g., changes in viral growth in culture, temperature sensitivity, plaque size, attenuation, and immunogenicity.
In one aspect of the invention, isolated polynucleotides and expression vectors are provided which comprise a chimeric RSV genome or antigenome having at least one attenuating point mutation adopted from a biologically derived mutant RSV. In one such embodiment, at least one point mutation is present in the polymerase gene L involving a nucleotide substitution that specifies a ts phenotype. Exemplary RSV clones and vectors incorporate a nucleotide substitution that results in an amino acid change in the polymerase gene at Phe521, Gln831, Met1169, or Tyr1321. Preferably, two or three mutations are incorporated in a codon specifying the attenuating mutation in order to increase the level of genetic stability. Other exemplary RSVs incorporate at least two attenuating ts mutations.
Mutations incorporated within chimeric cDNAs, vectors and viral particles of the invention can be introduced individually or in combination into a full-length RSV cDNA and the phenotypes of rescued virus containing the introduced mutations can be readily determined. In exemplary embodiments, amino acid changes displayed by attenuated, biologically-derived viruses versus a wild-type RSV, for example changes exhibited by cpRSV or tsRSV, are incorporated in combination within recombinant RSV to yield a desired level of attenuation.
The present invention also provides chimeric RSV clones, vectors and particles incorporating multiple, phenotype-specific mutations introduced in selected combinations into the chimeric genome or antigenome to produce an attenuated, infectious virus or subviral particle. This process, coupled with routine phenotypic evaluation, provides chimeric RSV having such desired characteristics as attenuation, temperature sensitivity, altered immunogenicity, cold-adaptation, small plaque size, host range restriction, etc. Mutations thus identified are compiled into a xe2x80x9cmenuxe2x80x9d and introduced in various combinations to calibrate a vaccine virus to a selected level of attenuation, immunogenicity and stability.
In preferred embodiments, the invention provides for supplementation of one or more mutations adopted from biologically derived RSV, e.g., cp and ts mutations, with additional types of mutations involving the same or different genes. Target genes for mutation in this context include the attachment (G) protein, fusion (F) protein, small hydrophobic (SH), RNA binding protein (N), phosphoprotein (P), the large polymerase protein (L), the transcription elongation factor (M2), M2 ORF2, the matrix (M) protein, and two nonstructural proteins, NS1 and NS2. Each of these proteins can be selectively deleted, substituted or rearranged, in whole or in part, alone or in combination with other desired modifications, to achieve novel chimeric RSV recombinants.
In one aspect, the SH gene is deleted in the donor or recipient context to yield a chimeric RSV having novel phenotypic characteristics, including enhanced growth in vitro and/or attenuation in vivo. In a related aspect, this gene deletion, or another selected, non-essential gene or gene segment deletion, such as a NS1 or NS2 gene deletion is combined in a chimeric RSV with one or more separate mutations specifying an attenuated phenotype, e.g., a point mutation adopted directly (or in modified form, e.g., by introducing multiple nucleotide changes in a codon specifying the mutation) from a biologically derived attenuated RSV mutant.
For example, the SH gene or NS2 gene may be deleted in combination with one or more cp and/or ts mutations adopted from cpts248/404, cpts530/1009, cpts530/1030, or another selected mutant RSV strain, to yield a chimeric RSV having increased yield of virus, enhanced attenuation, and genetic resistance to reversion from an attenuated phenotype, due to the combined effects of the different mutations.
In addition, a variety of other genetic alterations can be produced in a chimeric RSV genome or antigenome, alone or together with one or more attenuating point mutations adopted from a biologically derived mutant RSV. For example, genes or gene segments from non-RSV sources may be inserted in whole or in part. Alternatively, the order of genes can be changed, gene overlap removed, or a RSV genome promoter replaced with its antigenome counterpart. Different or additional modifications in the chimeric genome or antigenome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions (e.g., a unique Stul site between the G and F genes) or elsewhere. Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.
Alternatively, polynucleotide molecules or vectors encoding the chimeric RSV genome or antigenome can be modified to encode non-RSV sequences, e.g., a cytokine, a T-helper epitope, a restriction site marker, or a protein of a microbial pathogen (e.g., virus, bacterium or fungus) capable of eliciting a protective immune response in an intended host.
In other embodiments the invention provides a cell or cell-free lysate containing an expression vector which comprises an isolated polynucleotide molecule encoding a chimeric RSV genome or antigenome as described above, and an expression vector (the same or different vector) which comprises one or more isolated polynucleotide molecules encoding the N, P, L and RNA polymerase elongation factor proteins of RSV. Upon expression the genome or antigenome and N, P, L, and RNA polymerase elongation factor proteins combine to produce an infectious RSV viral or subviral particle.
Attenuated chimeric RSV of the invention is capable of eliciting a protective immune response in an infected human host, yet is sufficiently attenuated so as to not cause unacceptable symptoms of severe respiratory disease in the immunized host. The attenuated chimeric virus or subviral particle may be present in a cell culture supernatant, isolated from the culture, or partially or completely purified. The virus may also be lyophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.
The invention further provides novel vaccines comprising a physiologically acceptable carrier and/or adjuvant and an isolated attenuated chimeric RSV as described above. In one embodiment, the vaccine is comprised of chimeric RSV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications as described above. The vaccine can be formulated in a dose of 103 to 106 PFU of attenuated virus. The vaccine may comprise attenuated chimeric virus that elicits an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups. In this regard, chimeric RSV of the invention can individually elicit a monospecific immune response or a polyspecific immune response against multiple RSV strains or subgroups. Chimeric RSV can be combined in vaccine formulations with other chimeric RSV or non-chimeric RSV having different immunogenic characteristics for more effective protection against one or multiple RSV strains or subgroups.
In related aspects, the invention provides a method for stimulating the immune system of an individual to elicit an immune response against one or more strains or subgroups of RSV in a mammalian subject. The method comprises administering a formulation of an immunologically sufficient amount of an attenuated, chimeric RSV as described above in a physiologically acceptable carrier and/or adjuvant. In one embodiment, the immunogenic composition is a vaccine comprised of chimeric RSV having at least one, and preferably two or more attenuating mutations or other nucleotide modifications as described above. The vaccine can be formulated in a dose of 103 to 106 PFU of attenuated virus. The vaccine may comprise attenuated chimeric virus that elicits an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups. In this context, the chimeric RSV can elicit a monospecific immune response or a polyspecific immune response against multiple RSV strains or subgroups. Alternatively, chimeric RSV having different immunogenic characteristics can be combined in a vaccine mixture or administered separately in a coordinated treatment protocol to elicit more effective protection against one RSV strain, or against multiple RSV strains or subgroups.